Rapid Determination of Methotrexate and 7-Hydroxymethotrexate in Serum and Cerebrospinal Fluid by Radial Compression Liquid Chromatography

Abstract

A rapid, specific and sensitive radial compression reverse phase liquid chromatographic method for the analysis of methotrexate and 7-hydroxymethotrexate in serum and cerebrospinal fluid is reported. A mobile phase consisting of acetonitrile-methanol-pH 3 phosphate (8:15:77) at 6 ml/min flow rate was employed. The U.V. detector was set at 317 nm, and folic acid was used as an internal standard. A rapid extraction of methotrexate and 7-hydroxymethotrexate was performed using Sep-Pak cartridges with high extraction efficiency for both compounds. Patients serum and cerebrospinal fluid samples were analyzed by the described method and the concentrations of methotrexate were compared to those obtained by an enzyme immunoassay. No interference from other metabolites or anticancer drugs in the described assay was observed.